Molecular Cloning: A Laboratory Manual, Book 2 |
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Page 24
It is therefore important to arrange the ligation conditions to minimize the
formation of these chimeric molecules , since it is extremely laborious to test
subsequently whether cDNA clones that contain the particular restriction site at
an internal ...
It is therefore important to arrange the ligation conditions to minimize the
formation of these chimeric molecules , since it is extremely laborious to test
subsequently whether cDNA clones that contain the particular restriction site at
an internal ...
Page 24
GENERATION OF A LIBRARY OF RANDOMLY OVERLAPPING CLONES
Purification and Ligation of the Target DNA 1 . ... Because the next step of the
procedure requires the target DNA to be ligated to itself , it is best to use
restriction ...
GENERATION OF A LIBRARY OF RANDOMLY OVERLAPPING CLONES
Purification and Ligation of the Target DNA 1 . ... Because the next step of the
procedure requires the target DNA to be ligated to itself , it is best to use
restriction ...
Page 90
5 ug phosphorylated hexameric linkers ( 1 ug / ul ) 2 ul 10 x ligase buffer 1 ul H ,
O to 9 ul Add 2 Weiss units of bacteriophage T4 DNA ligase ... Heat the ligation
mixture to 68°C for 10 minutes to inactivate the bacteriophage T4 DNA ligase . iv .
5 ug phosphorylated hexameric linkers ( 1 ug / ul ) 2 ul 10 x ligase buffer 1 ul H ,
O to 9 ul Add 2 Weiss units of bacteriophage T4 DNA ligase ... Heat the ligation
mixture to 68°C for 10 minutes to inactivate the bacteriophage T4 DNA ligase . iv .
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Contents
TEST LIGATION 3 29 | 3 |
SYNTHESIS OF THE FIRST STRAND OF cDNA 8 | 11 |
FRAGMENT OF E coli DNA POLYMERASE I 13 59 | 14 |
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Common terms and phrases
acid activity agarose aliquots allow amount amplification appropriate approximately bacteriophage bacteriophage T4 base buffer carried cDNA cells centrifugation Chapter chromatography cleaved clones colonies concentration constructed containing deletions denatured described determine digestion DNA polymerase dNTPs double-stranded efficiency electrophoresis et al ethanol expression Figure filters fragment gene genomic hybridization increase Incubate inserted interest isolated labeled length libraries ligation linear linkers method microfuge minutes mixture molecules mRNA mutagenesis mutagenic mutations Nucleic nucleotides obtained oligonucleotide original plaques plasmid plasmid DNA plate pool position possible prepared presence primer probe problem protein purified radiolabeled reaction recombinant region remove restriction enzyme room temperature samples screening sequence single single-stranded solution specific step Store strand synthesis target DNA target sequence template termini tion transfer transformants tube units usually vector volume